Fourier-transform infrared study on effects of ageing, oestrogen level and altered dietary loading on rat mandibular condylar cartilage.

OBJECTIVE: Mandibular condylar cartilage (MCC) of the rat was examined with the Fourier-transform infrared (FITR) spectroscopic imaging to study the effects of ageing, oestrogen level and altered dietary loading on the structure of MCC. MATERIALS AND METHODS: The Sprague-Dawley rats (n = 96) aged 5 and 14 months were divided into 12 subgroups according to age, oestrogen status (ovariectomized [OVX], non-ovariectomized [non-OVX)]) and diet (hard, normal, soft). Specimens of the MCC were examined with FTIR spectroscopic imaging to quantify the distribution of collagens and proteoglycans. MCC was divided sagittally into three segments: anterior, most superior and posterior. From each segment, the collagen and proteoglycan contents at different depths of cartilage were statistically compared between the groups using an N-way analysis of variance (ANOVA). RESULTS: The amount of collagen content was significantly associated with old age in the deep layer of the anterior segment and in the middle layer of the posterior segment of MCC. In the deep layer of the most superior segment, the collagen content also increased with ageing. The amount of proteoglycan content increased significantly when dietary loading increased, and the oestrogen level decreased in the deep layer of the most superior segment of MCC. CONCLUSION: Ageing, oestrogen level and altered dietary loading have a significant effect on the location and content of collagens and proteoglycans of rat MCC. Ageing significantly increased the amount of collagen content in the superior and posterior segments, being highest in the older soft-diet rats. Decreased oestrogen levels and increased dietary loading increased the amount of proteoglycan content.

Long-term haplodeficency of DSPP causes temporomandibular joint osteoarthritis in mice.

BACKGROUND: Extracellular matrix (ECM) protein malfunction or defect may lead to temporomandibular joint osteoarthritis (TMJ OA). Dentin sialophophoprotein (DSPP) is a mandibular condylar cartilage ECM protein, and its deletion impacted cell proliferation and other extracellular matrix alterations of postnatal condylar cartilage. However, it remains unclear if long-term loss of function of DSPP leads to TMJ OA. The study aimed to test the hypothesis that long-term haploinsufficiency of DSPP causes TMJ OA. MATERIALS AND METHODS: To determine whether Dspp+/- mice exhibit TMJ OA but no severe tooth defects, mandibles of wild-type (WT), Dspp+/-, and Dspp homozygous (Dspp-/-) mice were analyzed by Micro-computed tomography (micro-CT). To characterize the progression and possible mechanisms of osteoarthritic degeneration over time in Dspp+/- mice over time, condyles of Dspp+/- and WT mice were analyzed radiologically, histologically, and immunohistochemically. RESULTS: Micro-CT and histomorphometric analyses revealed that Dspp+/- and Dspp-/- mice had significantly lower subchondral bone mass, bone volume fraction, bone mineral density, and trabecular thickness compared to WT mice at 12 months. Interestingly, in contrast to Dspp-/- mice which exhibited tooth loss, Dspp+/- mice had minor tooth defects. RNA sequencing data showed that haplodeficency of DSPP affects the biological process of ossification and osteoclast differentiation. Additionally, histological analysis showed that Dspp+/- mice had condylar cartilage fissures, reduced cartilage thickness, decreased articular cell numbers and severe subchondral bone cavities, and with signs that were exaggerated with age. Radiographic data showed an increase in subchondral osteoporosis up to 18 months and osteophyte formation at 21 months. Moreover, Dspp+/- mice showed increased distribution of osteoclasts in the subchondral bone and increased expression of MMP2, IL-6, FN-1, and TLR4 in the mandibular condylar cartilage. CONCLUSIONS: Dspp+/- mice exhibit TMJ OA in a time-dependent manner, with lesions in the mandibular condyle attributed to hypomineralization of subchondral bone and breakdown of the mandibular condylar cartilage, accompanied by upregulation of inflammatory markers.

280 mT static magnetic field promotes the growth of postpartum condylar cartilage.

PURPOSE: Functional appliances made of permanent magnets have been used in jaw orthopedic treatment. However, whether the static magnetic field (SMF) generated by permanent magnets promotes the developmental sequence of condylar cartilage and thus promotes the growth of the mandible remains to be studied. The aim of this study was to investigate the effects of 280 mT SMF on postnatal condylar chondrogenesis and endochondral ossification and the roles of FLRT3, FGF2 and BMP2 signaling in this chondrodevelopmental sequences. METHODS: Forty-eight rats were assigned to two groups (control and SMF). The condyles were collected at the specified time points. The histomorphological changes in the condyle were observed by histological staining. The expression of proteins related to the proliferation and differentiation of the condylar cartilage and the changes in subchondral bone microstructure were analyzed by immunohistochemical staining and micro-CT scanning. FLRT3, FGF2, and BMP2 expression was detected by immunofluorescence staining. RESULTS: Under SMF stimulation, the cartilage of young rats grew longitudinally and laterally, and the thickness of the cartilage became thinner as it grew. The SMF promoted the proliferation and differentiation of condylar chondrocytes and endochondral ossification and increased subchondral bone mineral density, and BMP2 signaling was involved. Moreover, under SMF loading, the increased expression of FGF2 and FLRT3 were involved in regulating cartilage morphogenesis and growth. In late development, the decreased expression of FGF2/FLRT3 and the increased expression of BMP2 promoted endochondral ossification. The SMF accelerated this opposite expression trend. CONCLUSION: FGF2/FLRT3 and BMP2 signals are involved in the regulatory effect of SMF exposure on chondrogenesis and endochondral ossification, which provides a theoretical basis for the clinical use of magnetic appliances to promote condylar growth.

Age-Related Gene and Protein Expression in Mouse Mandibular Condyle Analyzed by Cap Analysis of Gene Expression and Immunohistochemistry.

INTRODUCTION: Aging, an inevitable physiological process, leads to morphological and histological degenerative changes in the mandibular condylar cartilage (MCC); however, the molecular mechanism has not yet been elucidated, and little information is available on age-related factors. Therefore, this study was designed to identify age-related factors by investigating the age-related differentially expressed genes (DEGs) and localization of their translated protein expression in the mandibular condyle. METHODS: Mandibular condyles were collected from 10- and 50-week-old mice. Total RNA was extracted from the samples and then analyzed using cap analysis of gene expression (CAGE) to identify age-related DEGs. Gene ontology (GO) enrichment analysis was performed to determine which biological processes were most affected by aging in terms of gene expression using Metascape. The mandibular condyle samples were processed for histology to investigate morphological changes caused by aging and for immunohistochemistry to localize the protein expression encoded by age-related genes identified with CAGE. Semi-quantitative immunohistochemistry was performed to assess age-related extracellular matrix (ECM) protein levels in the MCC. The histological sections were also used for Alcian blue histochemistry to detect glycosaminoglycans (GAGs). RESULTS: GO enrichment analysis revealed that the genes related to "extracellular matrix organization," including Acan, Col1a1, Col1a2, Col2a1, Mmp3, Mmp9, and Mmp13, were most differentially expressed in the aged mandibular condyle. Among these seven genes, Mmp3 was upregulated, and the others were downregulated with aging. Histological examination showed the age-related morphological and histological changes in the MCC. Immunohistochemical investigation showed the localization of matrix metalloproteinases (MMPs)-3, -9, and -13 and their substrate proteins, aggrecan, type I collagen, and type II collagen, in the mandibular condyle at 10 and 50 weeks, indicating different localizations between the young and the aged. In the aged MCC, semi-quantitative immunohistochemistry showed a significant decrease in the aggrecan protein level, and Alcian blue histochemistry showed a decrease in GAGs. CONCLUSION: MMP-3, MMP-9, and MMP-13 contribute to the remodeling of the ECM of the MCC and subchondral bone during aging by degrading ECM proteins at specific times and sites under the regulation of their production and secretion.

Mechanism and potential contributing factors to temporomandibular joint osteoarthritis.

OBJECTIVE: To investigate the mechanism of and potential contributing factors to temporomandibular joint osteoarthritis (TMJOA) caused by oestrogen deficiency with a persistent high bite force. MATERIALS AND METHODS: A TMJOA model was generated by subjecting 6-week-old female rats to ovariectomy (OVX) and feeding them a hard feed. The rats (n = 12/group) were divided into sham (control); OVX; OVX+hard feed (HF); OVX+hard feed+local-joint injection of 17ß-oestradiol (an oestrogen) (E2); and OVX+hard feed+local-joint injection of rapamycin (an autophagy activator) (RAPA)groups. Condyles were stained with haematoxylin-eosin and Safranin O Fast Green. The expression of Beclin 1, LC3 and p-mTOR in condylar cartilages was analysed. RESULTS: Tissue staining revealed thinner condylar cartilage, varying numbers or fewer hypertrophic chondrocytes, and lower proteoglycan content in the cartilage matrix of the OVX group. These characteristics were more pronounced in the HF group, but were significantly recovered in the E2 and RAPA groups. Immunohistochemical staining revealed significantly lower autophagic flux in OVX/HF groups and a higher one in E2/RAPA groups. CONCLUSIONS: A persistent high bite force could aggravate TMJOA induced by oestrogen deficiency, and the application of oestrogen or rapamycin could delay its progression. Additionally, autophagy may play a role in the development of TMJOA.

Role of HIF-2α/NF-κB pathway in mechanical stress-induced temporomandibular joint osteoarthritis.

OBJECTIVES: Many activities overload temporomandibular joint (TMJ) and cause mandibular condylar cartilage (MCC) degradation by inducing the expression of hypoxia-inducible factor-2α (HIF-2α). Although NF-κB signaling pathway has been reported to induce HIF-2α expression, the underlying mechanisms need to be verified. The aim was to investigate the effects of NF-κB/HIF-2α on MCC degradation induced by mechanical stress and the regulatory mechanism of NF-κB in the HIF-2α pathway. METHODS: Chondrocytes were subjected to cyclic compressive forces in a hypoxic environment. Western blotting was used to test the effects of stress on the expression of NF-κB and HIF-2α. HIF-2α siRNA and shRNA were constructed and transfected into MCC cells in vitro and in vivo to inhibit HIF-2α expression. To test the regulatory effect of the NF-κB pathway on HIF-2α, siRNA p65 was transfected into MCC. RESULTS: The results showed that mechanical stress could cause cartilage degradation and significantly increased the expression of NF-κB, HIF-2α, and downstream degradation factors (MMP13 and ADAMTs-4). Blockade of HIF-2α decreased cartilage degradation and related degradation factors. Suppression of p65 significantly decreased the expression of HIF-2α. CONCLUSIONS: Our results indicated that the upstream NF-κB pathway exerted a regulatory effect on HIF-2α in the degradation of MCC induced by stress.

Mechanoresponsive and lubricating changes of mandibular condylar cartilage associated with mandibular lateral shift and recovery in the growing rat.

OBJECTIVE: The in vivo mechanoresponsive and lubricating changes of the mandibular condylar cartilage (MCC) associated with mandibular lateral shift (MLS) and recovery are poorly understood. Using growing rats, we investigated whether the expression of mechanoresponsive factors, including proteoglycan-4 (PRG4), Indian hedgehog (Ihh) and transforming growth factor-ß1 (TGF-ß1), would be affected by MLS. We also investigated whether these changes could recover to the control level after a 2-week treatment reversal (TR). MATERIALS AND METHODS: The MLS appliances were placed for 2 or 4 weeks in 5-week-old rats and removed from 7-week-old rats in the TR group. The MCC was analysed histomorphometrically by toluidine blue staining. Reverse transcription-polymerase chain reaction and immunohistochemistry were performed to evaluate the expression of PRG4, Ihh, PTHrP (parathyroid hormone-related protein), TGF-ß1, Matrix metallopeptidase 13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5). RESULTS: A thickened superficial layer and an enhanced expression of PRG4 were detected in MLS groups. PTHrP-Ihh expression correlated positively with the up-regulation of PRG4. TGF-ß1 expression decreased in the early stage of MLS but recovered to the control level in the TR group. A significantly enhanced expression of MMP-13 in MLS groups was detected. CONCLUSION: MLS treatment, which acted on the growth stage of rats, affected the morphology and expression of lubrication factor in the MCC. Elimination of this mechanical stimulus may help MCC recover to normal conditions. CLINICAL RELEVANCE: Our study supports that the adaptive changes of MCC, which are caused by mandibular functional deviation, could be largely recovered by early treatment.

Temporomandibular Joint Hypofunction Secondary to Unilateral Partial Discectomy Attenuates Degeneration in Murine Mandibular Condylar Cartilage.

Mechanical overloading of the temporomandibular joint (TMJ) promotes both the initiation and progression of TMJ osteoarthritis (OA). New preclinical animal models are needed for the evaluation of the molecular basis of cellular load transmission. This would allow a better understanding of the underlying mechanisms of TMJ-OA pain and disability, and help identify new therapeutics for its early diagnosis and management. The purpose of this study was to evaluate the role of mechanical loading in the progression of TMJ-OA in surgical instability arising from unilateral partial discectomy (UPD) in a murine model. In the theoretical modelling employed, lower joint reaction forces were observed on the chewing (working) side of the TMJ in the murine craniomandibular musculoskeletal system. Hypofunction was induced secondary to UPD through surgically manipulating the working side using an unopposed molar model. When the working side was restricted to the same side as that on which UPD was performed, late-stage degeneration of the cartilage showed a significant reduction (p<0.05), with diminished fibrillation and erosion of the articular cartilage, cell clustering, and hypocellularity. Condylar remodelling and proteolysis of proteoglycans were less affected. Thus, select and specific late-stage changes in TMJ-OA were contextually linked with the local mechanical environment of the joint. These data underscore the value of the UPD mouse model in studying mechanobiological pathways activated during TMJ-OA, and suggest that therapeutically targeting mechanobiological stimuli is an effective strategy in improving long-term biological, clinical, and patient-based outcomes.

ADAMTSL1 and mandibular prognathism.

Mandibular prognathism is characterized by a prognathic or prominent mandible. The objective of this study was to find the gene responsible for mandibular prognathism. Whole exome sequencing analysis of a Thai family (family 1) identified the ADAMTSL1 c.176C>A variant as the potential defect. We cross-checked our exome data of 215 people for rare variants in ADAMTSL1 and found that the c.670C>G variant was associated with mandibular prognathism in families 2 and 4. Mutation analysis of ADAMTSL1 in 79 unrelated patients revealed the c.670C>G variant was also found in family 3. We hypothesize that mutations in ADAMTSL1 cause failure to cleave aggrecan in the condylar cartilage, and that leads to overgrowth of the mandible. Adamtsl1 is strongly expressed in the condensed mesenchymal cells of the mouse condyle, but not at the cartilage of the long bones. This explains why the patients with ADAMTSL1 mutations had abnormal mandibles but normal long bones. This is the first report that mutations in ADAMTSL1 are responsible for the pathogenesis of mandibular prognathism.

Effects of loading on chondrocyte hypoxia, HIF-1α and VEGF in the mandibular condylar cartilage of young rats.

OBJECTIVES: To investigate hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) expression under altered loading, and to explore the relationship between loading and hypoxia in the mandibular condylar cartilage of young rats. SETTING AND SAMPLE POPULATION: Eighty Sprague-Dawley rats. MATERIAL AND METHODS: The reduced loading group was fed soft food, and their incisors were cut to avoid occlusal contact. The increased loading group was fed hard food and had forced jaw-opening. Ten rats from each group (n = 10) were sacrificed at 12, 24, 48, and 96 hours after initiation of the experiment. Pimonidazole hydrochloride (Hypoxyprobe-1, HP-1) was used as a hypoxia marker to confirm the hypoxic state. Hypoxic chondrocytes as indicated by HP-1, HIF-1α and VEGF protein expressions were recognized by immunohistochemical detection. HIF-1α and VEGF mRNA expressions were detected by semi-quantitative RT-PCR. RESULTS: Hypoxyprobe-1 was confined in the upper layers of cartilage, and was most strongly expressed in the weight-bearing area of TMJ at 12 and 96 hours. Staining of HIF-1α and VEGF was most strongly expressed in the chondrocytes of the fibrous and proliferative layer at all time points. Furthermore, expressions were also displayed in the hypertrophic and calcified layers at 48 and 96 hours. The expressions of HIF-1α and VEGF mRNA were higher in the increased loading group than in the reduced loading group at 48 and 96 hours (P < . 05). CONCLUSION: Mechanical loading seems to directly induce weight-bearing area hypoxia followed by new vessel formation, which indicates that these factors are related and important for the development of cartilage.

Sex differences in the estrogen-dependent regulation of temporomandibular joint remodeling in altered loading.

OBJECTIVE: Temporomandibular joint (TMJ) diseases predominantly afflict women, suggesting a role of estrogen in the disease etiology. Previously, we determined that decreased occlusal loading (DOL) inhibited collagen type II (Col2) expression in the mandibular condylar cartilage (MCC) of female wild-type (WT) mice whereas no change was observed in males. This decrease in chondrogenesis was abolished by estrogen receptor beta (ERß) deficiency in females. Therefore, the goal of this study was to examine the role of estradiol - ERß signaling in mediating DOL effects in male mice to further decipher sex differences. METHODS: Male 21 day-old WT and ERßKO male mice were treated with either placebo or estradiol and exposed to normal or DOL for 4 weeks. Cartilage thickness and cell proliferation, gene expression and immunohistochemistry of chondrogenic markers and estrogen receptor alpha (ERα), and analysis of bone histomorphometry via microCT were completed to ascertain the effect of estradiol on DOL effects to the TMJ. RESULTS: ERßKO male mice lack a MCC phenotype. In both genotypes, estradiol treatment increased Col2 gene expression and trabecular thickness. DOL in combination with estradiol treatment caused a significant increase in Col2 gene expression in both genotypes. CONCLUSIONS: The sex differences in DOL-induced inhibition of Col2 expression do not appear to be mediated by differences in estradiol levels between male and female mice. Greater understanding on the role of estrogen and altered loading are critical in order to decipher the sex dimorphism of TMJ disorders.

PTH [1-34]-induced alterations predispose the mandibular condylar cartilage to mineralization.

OBJECTIVE: To study the effects of intermittent parathyroid hormone (PTH [1-34]) on the mandibular condylar cartilage (MCC) and subchondral bone in adult female mice. MATERIALS AND METHODS: Twenty-two, 20-week-old female mice were used for in vivo experiments. The experimental mice (n=11) received daily intraperitoneal injections of PTH [1-34] for 3 weeks, while control mice (n=11) received intraperitoneal injections of 0.9% saline solution. Mice were euthanized and then micro-computed tomography (micro-CT); histology and immunostaining were carried out to assess the response. RESULTS: Intermittent PTH [1-34] led to early MCC breakdown and surface irregularities. Micro-CT analyses indicated that PTH [1-34] treatment led to increased bone volume fraction, tissue density and trabecular thickness, while decreasing the trabecular spacing. Histological analyses showed decreased proteoglycan secretion, increased bone turnover (TRAP staining) and increased mineralization. Furthermore, PTH [1-34] treatment showed increased apoptosis of the cells. Our immunohistochemistry showed increased expression of pSMAD158 in the MCC and subchondral bone with PTH administration, whereas sclerostin (SOST) expression was decreased. CONCLUSIONS: Intermittent PTH [1-34] results in early mineralization of the MCC, which may result in cartilage degeneration. Our results identified a novel mechanism by which PTH [1-34] induces alteration in the microarchitecture of the MCC and the subchondral bone.

Estrogen receptor alpha mediates mandibular condylar cartilage growth in male mice.

OBJECTIVES: In the appendicular skeleton, estrogen via ERα signalling has been shown to mediate endochondral growth plate fusion in both males and females. However, the role of ERα in mediating growth of the mandibular condylar cartilage is unknown. Thus, this study focuses on the characterization of the mandibular condylar cartilage phenotype in young and adult male ERαKO mice. SETTING: Columbia University Medical Center. MATERIAL AND METHODS: WT and ERαKO C57BL/6 male mice were sacrificed at 49 days or 9 months for phenotypic analysis. Changes to MCC thickness, cell number and cell density were measured using histomorphometric methods. Cartilage-specific gene expression and OARSI scores were investigated for 49-day and 9-month-old male ERαKO and WT mice. RESULTS: In young mice, a significant increase in the number of mandibular condylar cartilage cells and a significant decrease in the expression of Col10, Runx2 and DMP1 were observed in the male ERαKO mice compared to WT. In 9-month-old mice, we found a similar increase in the number of cells but no change in osteoarthritic histological scoring in ERαKO mice compared to WT mice. CONCLUSION: In summary, estrogen plays a role in mediating mandibular condylar maturation in young male mice. However, according to this study, it does not play a role in mediating long-term growth or age-related mandibular condylar cartilage degeneration in males.

Diverse contribution of Col2a1-expressing cells to the craniofacial skeletal cell lineages.

OBJECTIVES: Craniofacial skeletal development requires deliberate coordination of two distinct mechanisms of endochondral and intramembranous ossification. Col2a1-expressing cells encompass growth-associated skeletal progenitors in endochondral bones of the limb. The objective of this study was to determine the contribution of Col2a1-expressing cells to the craniofacial skeletal cell lineages. We hypothesize that Col2a1-expressing progenitors significantly contribute to various modes of ossification associated with the craniofacial development. METHODS: Cellular fates of Col2a1-expressing cells were studied based on a cre-loxP system using a Col2a1-cre transgene and an R26R-tdTomato reporter allele. We analysed three distinct locations of the craniofacial skeletal complex representing unique ossification mechanisms: the cranial base, the calvaria and the mandibular condyle. RESULTS: Col2a1-cre consistently marked a majority of skeletal cells in the cranial base. Interestingly, Col2a1-cre also marked a large number of osteoblasts and suture mesenchymal cells in the calvaria, in addition to chondrocytes in the underlying transient cartilage. In the mandibular condyle, Col2a1-cre marked chondrocytes and osteoblasts only during the growth phase. CONCLUSIONS: Col2a1 is expressed by progenitors of the skeletal lineage in canonical endochondral bone formation occurring in the cranial base. In contrast, other ossification mechanisms of the craniofacial complex utilize Col2a1-expressing cells in a different manner, whereby Col2a1 may be expressed in more differentiated or transient cell types of the skeletal lineage.

Genes that regulate morphogenesis and growth of the temporomandibular joint: a review.

Compared with the joints of the limbs, our understanding of the genes that regulate development and growth in the temporomandibular joint (TMJ) is fairly limited. Because the morphogenesis of the secondary cartilage and other intra-articular structures in the TMJ occurs later and in a different manner than in the limbs, the genetic control of TMJ development might reasonably be assumed to differ from that in the limbs. However, studies of the specific genes regulating TMJ morphogenesis and growth have only begun to appear in the literature within the last decade. This review attempts to survey and interpret the existing knowledge on this topic and to suggest fruitful avenues of investigation for the future. Studies to date using knockout and over-expression of candidate genes suggest that a developmental hierarchy of joint structures exists, with condyle development primary. A hierarchy of gene expression also exists: Runx2 and Sox9 expression is critical for condylar cartilage formation. Several of the other genes discussed in this report may regulate TMJ morphogenesis by affecting Sox9 and Runx2 expression and control the ihh-PTHrP axis by means of these genes.

Critical role of Bmpr1a in mandibular condyle growth.

The importance of Bone Morphogenetic Proteins (BMPs) in the regulation of cell fate, differentiation and proliferation in the growth plate is well-known. However, in secondary cartilages (such as that in the temporomandibular joint) that grow by proliferation of prechondrocytes and differ in their pattern of growth, the role of BMPs is largely unexplored. To examine this question, we ablated Bmpr1a in the condylar cartilage of neonatal mice and assessed the consequences for mandibular condyle growth and organization at intervals over the ensuing 4 weeks. Bmpr1a deficiency caused significant chondrodysplasia and almost eliminated the chondrocytic phenotype in the TMJ. Expression of Sox9, collagen II, proteoglycan were all greatly reduced, and cell proliferation as detected by BrdU was almost non-existent in the knockout mice. Primary bone spongiosa formation was also disturbed and was accompanied by reduced Osterix expression. These findings strongly suggest that Bmpr1a is critical for the development and growth of the mandibular condyle via its effect on proliferation of prechondroblasts and chondrocyte differentiation.

Critical signaling molecules in the temporomandibular joint osteoarthritis under different magnitudes of mechanical stimulation.

The mechanical stress environment in the temporomandibular joint (TMJ) is constantly changing due to daily mandibular movements. Therefore, TMJ tissues, such as condylar cartilage, the synovial membrane and discs, are influenced by different magnitudes of mechanical stimulation. Moderate mechanical stimulation is beneficial for maintaining homeostasis, whereas abnormal mechanical stimulation leads to degeneration and ultimately contributes to the development of temporomandibular joint osteoarthritis (TMJOA), which involves changes in critical signaling molecules. Under abnormal mechanical stimulation, compensatory molecules may prevent degenerative changes while decompensatory molecules aggravate. In this review, we summarize the critical signaling molecules that are stimulated by moderate or abnormal mechanical loading in TMJ tissues, mainly in condylar cartilage. Furthermore, we classify abnormal mechanical stimulation-induced molecules into compensatory or decompensatory molecules. Our aim is to understand the pathophysiological mechanism of TMJ dysfunction more deeply in the ever-changing mechanical environment, and then provide new ideas for discovering effective diagnostic and therapeutic targets in TMJOA.

Alendronate treatment rescues the effects of compressive loading of TMJ in osteogenesis imperfecta mice.

BACKGROUND: Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue caused by mutations associated with type I collagen, which results in defective extracellular matrix in temporomandibular joint (TMJ) cartilage and subchondral bone. TMJ is a fibrocartilaginous joint expressing type I collagen both in the cartilage and the subchondral bone. In the present study the effects of alendronate and altered loading of the TMJ was analyzed both in male and female OI mice. MATERIALS AND METHODS: Forty-eight, 10-weeks-old male and female OI mice were divided into 3 groups: (1) Control group: unloaded group, (2) Saline + Loaded: Saline was injected for 2 weeks and then TMJ of mice was loaded for 5 days, (3) alendronate + loaded: alendronate was injected for 2 weeks and then TMJ of mice was loaded for 5 days. Mice in all the groups were euthanized 24-h after the final loading. RESULTS: Alendronate pretreatment led to significant increase in bone volume and tissue density. Histomorphometrically, alendronate treatment led to increase in mineralization, cartilage thickness and proteoglycan distribution. Increased mineralization paralleled decreased osteoclastic activity. Our immunohistochemistry revealed decreased expression of matrix metallopeptidase 13 and ADAM metallopeptidase with thrombospondin type 1 motif 5. CONCLUSION: The findings of this research support that alendronate prevented the detrimental effects of loading on the extracellular matrix of the TMJ cartilage and subchondral bone.

Inflammation Triggers Chondrocyte Ferroptosis in TMJOA via HIF-1α/TFRC.

Inflammation and loss of articular cartilage are considered the major cause of temporomandibular joint osteoarthritis (TMJOA), a painful condition of the temporomandibular joint (TMJ). To determine the cause of TMJ osteoarthritis in these patients, synovial fluid of TMJOA patients was compared prior to and after hyaluronic lavage, revealing substantially elevated levels of interleukin (IL) 1ß, reactive oxidative stress (ROS), and an overload of Fe3+ and Fe2+ prior to lavage, indicative of ferroptosis as a mode of chondrocyte cell death. To ask whether prolonged inflammatory conditions resulted in ferroptosis-like transformation in vitro, we subjected TMJ chondrocytes to IL-1ß treatment, resulting in a shift in messenger RNA sequencing gene ontologies related to iron homeostasis and oxidative stress-related cell death. Exposure to rat unilateral anterior crossbite conditions resulted in reduced COL2A1 expression, fewer chondrocytes, glutathione peroxidase 4 (GPX4) downregulation, and 4-hydroxynonenal (4-HNE) upregulation, an effect that was reversed after intra-articular injections of the ferroptosis inhibitor ferrostatin 1 (Fer-1). Our study demonstrated that ferroptosis conditions affected mitochondrial structure and function, while the inhibitor Fer-1 restored mitochondrial structure and the inhibition of hypoxia-inducible factor 1α (HIF-1α) or the transferrin receptor 1 (TFRC) rescued IL-1ß-induced loss of mitochondrial membrane potential. Inhibition of HIF-1α downregulated IL-1ß-induced TFRC expression, while inhibition of TFRC did not downregulate IL-1ß-induced HIF-1α expression in chondrocytes. Moreover, inhibition of HIF-1α or TFRC downregulated the IL-1ß-induced MMP13 expression in chondrocytes, while inhibition of HIF-1α or TFRC rescued IL-1ß-inhibited COL2A1 expression in chondrocytes. Furthermore, upregulation of TFRC promoted Fe2+ entry into chondrocytes, inducing the Fenton reaction and lipid peroxidation, which in turn caused ferroptosis, a disruption in chondrocyte functions, and an exacerbation of condylar cartilage degeneration. Together, these findings illustrate the far-reaching effects of chondrocyte ferroptosis in TMJOA as a mechanism causing chondrocyte death through iron overload, oxidative stress, and articular cartilage degeneration and a potential major cause of TMJOA.

A PTHrP Gradient Drives Mandibular Condylar Chondrogenesis via Runx2.

The mandibular condylar cartilage (MCC) is an essential component of the temporomandibular joint, which orchestrates the vertical growth of the mandibular ramus through endochondral ossification with distinctive modes of cell differentiation. Parathyroid hormone-related protein (PTHrP) is a master regulator of chondrogenesis; in the long bone epiphyseal growth plate, PTHrP expressed by resting zone chondrocytes promotes chondrocyte proliferation in the adjacent layer. However, how PTHrP regulates chondrogenesis in the MCC remains largely unclear. In this study, we used a Pthrp-mCherry knock-in reporter strain to map the localization of PTHrP+ cells in the MCC and define the function of PTHrP in the growing mandibular condyle. In the postnatal MCC of PthrpmCherry/+ mice, PTHrP-mCherry was specifically expressed by cells in the superficial layer immediately adjacent to RUNX2-expressing cells in the polymorphic layer. PTHrP ligands diffused across the polymorphic and chondrocyte layers where its cognate receptor PTH1R was abundantly expressed. We further analyzed the mandibular condyle of PthrpmCherry/mCherry mice lacking functional PTHrP protein (PTHrP-KO). At embryonic day (E) 18.5, the condylar process and MCC were significantly truncated in the PTHrP-KO mandible, which was associated with a significant reduction in cell proliferation across the polymorphic layer and a loss of SOX9+ cells in the chondrocyte layers. The PTHrP-KO MCC showed a transient increase in the number of Col10a1+ hypertrophic chondrocytes at E15.5, followed by a significant loss of these cells at E18.5, indicating that superficial layer-derived PTHrP prevents premature chondrocyte exhaustion in the MCC. The expression of Runx2, but not Sp7, was significantly reduced in the polymorphic layer of the PTHrP-KO MCC. Therefore, PTHrP released from cells in the superficial layer directly acts on cells in the polymorphic layer to promote proliferation of chondrocyte precursor cells and prevent their premature differentiation by maintaining Runx2 expression, revealing a unique PTHrP gradient-directed mechanism that regulates MCC chondrogenesis.